Probiotic bacteria such as Bacillus subtilis and Bacillus licheniformis are used in the animal feed industry as supplement to the diet. Their usage is related to the ability of bacillus to replace or reduce the use of antibiotics, which are used as growth promoters in the animal feed industry.
Christian Hansen A/S, Denmark commercializes an example of such a probiotic growth-promoting product under the trade name GalliPro® (deposited as DSM 17231). GalliPro® is a Bacillus subtilis spore cell composition.
Besides the suggested mode of actions (e.g. immune modulation, gut flora modifier) probiotic bacillus are able to produce many beneficial components, such as enzymes, which are excreted in the gastro intestinal tract (GIT) when used as animal feed supplement. Enzymes such as phytase are excreted and improve the digestion and better uptake of animal feed (higher digestibility). The diet (feed) is mostly composed of plant origin such as grains, corn, soybean, soy oil and amino acids. Overall these effects contribute to the production of cost effective animal products.
Probiotic bacillus are also able to produce other beneficial components such as essential amino acids.
Bacillus spores can pass the acidic gastric barrier and germinate and outgrow within the gastrointestinal (GIT) of the animals. This has great advantages, since when ingested they can excrete numerous types of beneficial components, e.g. bacteriocins and also excrete useful essential amino acids. Moreover, the bacillus spores are thermostabile during a feed pelletizing process and are thereby an excellent delivery system to get both bacteriocins and e.g. essential amino acids into the GIT.
In the survival and proliferation process of bacillus in GIT, the role of bile is important. Bile is produced in the liver and stored in the gallbladder. Bile contains water, lecithin, bilirubin and biliverdin and bile salts.
It is known from the literature that bile has some negative influences on the survival and germination and outgrowth of bacillus spore cells to vegetative cells in the GIT of animals. Therefore research is ongoing to find probiotic bile resistant Bacillus strains.
The article (Antonie Van Leeuwenhoek. August 2006; 90(2): 139-46. Epub Jul. 4, 2006) describes isolation of a number of Bacillus samples/cell directly from the intestine of chickens. The isolated bacillus cells were tested for probiotic activity. The six bacilli with highest probiotic activity were testes for bile salt resistance and it was found that a specific highly probiotic bacillus has a relatively high level of bile salt resistance.
In this article there is no special focus on any time periods for the testing of bile resistance. In the experimental part the bacillus spore cells are simply tested for resistance after 5 days of presence in bile salt (see paragraph “Simulated small intestinal fluid tolerance test” on page 141).
US2003/0124104A describes that probiotic conventional bacillus endospores are sensitive to low concentration of bile salts, i.e. spore germination and/or rehydration is inhibited by the presence of even low concentrations of bile salts. This is contrary to other bacteria such as enteric pathogens, such as E. coli or S. aureus (see section [0014] to [0015]). In view of this it is suggested to screen/select for bacillus spores that are resistant to the inhibitory activity of bile salts, and as a result, germinate into vegetative cells, which then colonize the colon (see [0019]).
The working examples are all in presence and no real experimental data of actually screened specific Bacillus cell are provided in the description.
Further the bile salt screening conditions are relatively generically described. In particular there are no indications of any time periods for the selections of bile resistance. Said in other words, based on the only broad/generic teaching of this document one may select Bacillus cells that only can outgrow (germinate) slowly, i.e. are capable of germinating from spores to vegetative cells after e.g. 20 hours in presence of relevant amount of bile salt.
In this document there is no description or suggestion to select for bacillus cells that can outgrow (germinate) rapidly, i.e. capable of germinating and outgrowing from spores to vegetative cells reaching a defined growth point within a certain time interval in presence of a relevant amount of bile salt.
In summary, the prior art references relating to selection/screening of bile resistant bacillus cells are not focusing on rapid outgrowth/germination from spore cells to vegetative bacillus cells.
International PCT application with application number PCT/EP2008/057296 was filed Nov. 6, 2008. Applicant is Chr. Hansen A/S and it was NOT PUBLISHED at the filing date of this present application.
PCT/EP2008/057296 describes novel bacillus spores characterized by having an improved/rapid speed of germination and outgrowth from spore to vegetative cell in presence of a bile salt medium.
The bacillus spores as described herein have the same improved/rapid speed of germination and outgrowth from spore to vegetative cell as described in PCT/EP2008/057296.
PCT/EP2008/057296 only describes bacillus vegetative cells that are producing phytase in an increased amount as compared to the reference bacillus cell DSM 19467. There is NOT described not suggested to screen for a bacillus vegetative cell that produces essential amino acids with an increased amount as compared to the reference bacillus cell DSM 19467.
When there below is referred to prior art this shall be understood as prior art made available to the public (e.g. published articles/patents) at the filing date of this present application.